Atomic force microscopy measurement of leukocyte-endothelial interaction

Area of Science:

• Cellular and Molecular Biology
• Immunology
• Biophysics

Background:

• Leukocyte adhesion to vascular endothelium initiates inflammatory disease pathogenesis.
• Understanding these interactions is key to developing anti-inflammatory therapies.

Purpose of the Study:

• To quantify the forces involved in monocytic cell (HL-60) adhesion to endothelial cells (HUVECs).
• To investigate the role of specific adhesion molecules and blocking agents in leukocyte-endothelial interactions and transmigration.

Main Methods:

• Real-time force measurements using Atomic Force Microscopy (AFM) to analyze HL-60 and HUVEC interactions.
• Utilized monoclonal antibodies against integrins, selectins, ICAM-1, VCAM-1, and a cyclic RGD peptide for inhibition studies.
• Assessed transmigration of HL-60 cells across HUVEC monolayers.

Main Results:

• HL-60 cell adhesion forces were measured in the 40-100 pN range, with heightened adhesion at HUVEC borders.
• Beta1-integrins significantly mediated adhesion, while selectins, ICAM-1, and VCAM-1 played partial roles.
• A cyclic RGD peptide potently inhibited HL-60 adhesion and transmigration by interfering with VLA-4-VCAM-1 binding.
• Anti-beta1 and anti-alphaVbeta3 antibodies inhibited transmigration.

Conclusions:

• Beta1-integrins are critical for leukocyte-endothelial adhesion and transmigration.
• AlphaVbeta3 integrins play a role in leukocyte transmigration.
• The cyclic RGD peptide demonstrates significant potential for blocking both leukocyte adhesion and transmigration.